Protein C is a serine protease and naturally occurring anti-coagulant that plays a role in the regulation of hemostasis by inactivating Factors Va and VIIIa in the coagulation cascade. Human protein C is made in vivo as a single polypeptide of 461 amino acids. This polypeptide undergoes multiple post-translational modifications including, 1) cleavage of a 42 amino acid signal sequence; 2) cleavage of lysine and arginine residues (positions 156 and 157) to make a 2-chain inactive precursor or zymogen (an 155 amino acid residue light chain attached via a disulfide bridge to a 262 amino acid residue heavy chain); 3) vitamin K-dependent carboxylation of nine glutamic acid residues located within the amino-terminal 45 residues (gla-domain); and, 4) carbohydrate attachment at four sites (one in the light chain and three in the heavy chain). Finally, the 2-chain zymogen may be activated by removal of a dodecapeptide at the N-terminus of the heavy chain, producing activated protein C (aPC) possessing greater enzymatic activity than the 2-chain zymogen.
Blood coagulation is a highly complex process regulated by the balance between pro-coagulant and anti-coagulant mechanisms. This balance determines a condition of either normal hemostasis or abnormal pathological thrombus generation and the progression, for example, of coronary thrombosis leading to acute coronary syndromes (ACS; e.g. unstable angina, myocardial infarction). Two major factors control this balance; the generation of fibrin and the activation and subsequent aggregation of platelets, both processes controlled by the generation of the enzyme thrombin, which occurs following activation of the clotting cascade. Thrombin, in complex with thrombomodulin, also functions as a potent anti-coagulant since it activates protein C zymogen to aPC, which in turn inhibits the generation of thrombin. Thus, through the feedback regulation of thrombin generation via the inactivation of Factors Va and VIIIa, aPC functions as perhaps the most important down-regulator of blood coagulation resulting in protection against thrombosis. In addition to anti-coagulation, aPC has anti-inflammatory effects and exerts profibrinolytic properties that facilitate clot lysis.
Arterial thrombosis occurs in ACS in response to endothelial injury, typically as a result of a disruption of lipid-rich plaque. The initial phases of this response involve platelet adhesion, activation, and assembly of various pro-coagulants at the site of injury and on the surfaces of activated platelets. The resultant elaboration of thrombin generation plays a critical role in the progression of thrombus formation: both by fibrin deposition, and by platelet activation, thus potentiating the activation of the coagulation system. Traditional (e.g. unfractionated heparin [UFH]) and current (e.g. low-molecular weight heparin [LMWH]) anti-coagulant therapies for ACS rely on the inhibition of thrombin and/or Factor Xa (e.g. the heparins inactivate both thrombin and Xa by dramatically stimulating their interaction with anti-thrombin-III). However, due to steric constraints, these agents are not as effective in inhibiting clot-bound Xa or thrombin. The ability of aPC to target and to irreversibly inactivate the clot-bound Xa/Va complex attenuates local thrombin generation and the progression of thrombosis. Thus, aPC provides an advantage compared to current inhibitors of thrombin or Xa since the effect of decreased thrombin generation will persist after concentrations of aPC have decayed.
The critical role of aPC in controlling hemostasis is also exemplified by the increased rate of thrombosis in heterozygous deficiency, protein C resistance (e.g., due to the common Factor V Leiden mutation) and the fatal outcome of untreated homozygous protein C deficiency. Plasma-derived and recombinantly produced aPC have been shown to be effective and safe anti-thrombotic agents in a variety of animal models of both venous and arterial thrombosis.
Protein C levels have also been shown to be abnormally low in the following diseases and conditions: disseminated intravascular coagulation (DIC)[Fourrier, et al., Chest 101:816-823, 1992], sepsis [Gerson, et al., Pediatrics 91:418-422, 1993], major trauma/major surgery [Thomas, et al., Am J. Surg. 158:491-494, 1989], burns [Lo, et al., Burns 20:186-187 (1994)], adult respiratory distress syndrome (ARDS)[Hasegawa, et al., Chest 105(1):268-277, 1994], and transplantations [Gordon, et al., Bone Marrow Trans. 11:61-65 (1993)]. In addition, there are numerous diseases with thrombotic abnormalities or complications that aPC may be useful in treating, such as: heparin-induced thrombocytopenia (HIT) [Phillips, et al., Annals of Pharmacotherapy 28: 43-45, 1994], sickle cell disease or thalassemia [Karayalcin, et al., The American Journal of Pediatric Hematology/Oncology 11(3):320-323, 1989], viral hemorrhagic fever [Lacy, et al., Advances in Pediatric Infectious Diseases 12:21-53, 1997], thrombotic thrombocytopenic purpura (TTP) and hemolytic uremic syndrome (HUS)[Moake, Seminars in Hematology 34(2):83-89, 1997]. In addition, aPC in combination with Bactericidal Permeability Increasing Protein (BPI) may be useful in the treatment of sepsis [Fisher, et al., Crit. Care Med. 22(4):553-558, 1994].
It is well established that platelet inhibition is efficacious in both prevention and treatment of thrombotic disease. However, the use of anti-platelet agents, such as aspirin, increase the risk of bleeding, which limits the dose of the agent and duration of treatment. The combination of aPC and anti-platelet agents results in a synergy that allows the reduction of the dosages of both aPC and the anti-platelet agent(s). The reduction of the dosages of the agents in combination therapy in turn results in reduced side effects such as increased bleeding often observed in combination anti-coagulant/anti-platelet therapy.
Various methods of obtaining protein C from plasma and producing protein C, aPC and protein C/aPC polypeptides through recombinant DNA technology are known in the art and have been described. See e.g., U.S. Pat. Nos. 4,775,624 and 5,358,932. Despite improvements in methods to produce aPC through recombinant DNA technology, aPC and polypeptides thereof are difficult and costly to produce.
Unlike the zymogen protein C, activated protein C has an extremely short half-life. A major reason for the short half-life is that blood levels of aPC are regulated by molecules known as serpins (Serine Protease Inhibitors), which covalently bind to aPC forming an inactive serpin/aPC complex. The serpin/aPC complexes are formed when aPC binds and proteolytically cleaves a reactive site loop within the serpin; upon cleavage, the serpin undergoes a conformational change irreversibly inactivating aPC. The serpin/aPC complex is then eliminated from the bloodstream via hepatic receptors for the serpin/aPC complex. As a result, aPC has a relatively short half-life compared to the zymogen; approximately 20 minutes for aPC versus approximately 10 hours for human protein C zymogen (Okajima, et al., Thromb Haemost 63(1):48-53, 1990).
Therefore, an aPC derivative exhibiting resistance to serpin inactivation, while maintaining the desirable biological activities of aPC (e.g., anti-coagulant, fibrinolytic, and anti-inflammatory activities), provides a compound that has an increased plasma half-life and is effectively more potent than the parent compound, requiring substantially reduced dosage levels for therapeutic applications. The potency advantages are especially important in disease states in which serpin levels are elevated.